Beyofast sybr green qpcr mix 2

Yeast RNA was extracted using the hot acidic phenol method, while mammalian RNA was prepared using RNAeasy Animal Total RNA Isolation Kit (Beyotime R0032). For nuclear and cytoplasmic fraction separation, 5×10⁶ mammalian cells were washed twice with ice-cold PBS, collected, resuspended in 500 μL hypotonic buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂) by pipetting several times, and incubated on ice for 15 minutes. 25 μL 10% NP-40 was then added, and the homogenate was vortexed for 10 seconds at highest setting before centrifugation at 3,000 rpm at 4°C. The supernatant contained the cytoplasmic fraction, while the pellet was the nuclear fraction.
Reverse transcription was performed using BeyoRT II cDNA First Strand Synthesis Kit (RNase H-) (Beyotime D7168) with gene-specific primers, followed by qPCR using BeyoFast SYBR Green qPCR Mix (2×) (Beyotime D7260) with the primers listed in Table B in S1 Text.

Total RNA was extracted with TRIzol and checked using a Nanodrop 2000 (Agilent Technologies, Santa Clara, CA, USA) in different concentration dose of Am580 at 4th days. Quantitative PCR was carried out with BeyoFast™ SYBR Green qPCR Mix (2 ×) (Beyotime, China) in a 20 μL final volume according to the manufacturer’s instructions. Samples were run for 40 cycles at default thermal cycling conditions for SYBR Real-Time PCR (stage 1: 1 cycle, 95 °C for 2 min; stage 2: 40 cycles, 95 °C for 15 s, 60 °C for 30 s, 95 °C for 15 s). The primer sequences were as follows: GAPDH forward, 5′-GTGGACCTGACCTGCCGTCT-3′; GAPDH reverse, 5′-GGAGGAGTGGGTGTCGCTGT-3′; KCNT1 forward, 5′-CCGGACCTTCGAGTTTGACG-3′; KCNT1 reverse, 5′-GTCGCTCATCTTGAAGCCG-3′. Each sample was performed routinely in triplicate. The relative expression levels were analyzed using the 2^-ΔΔCT method.